| Objective①To clarify the species structure characteristics of gut microbiota in db/db mice and Jiangtang Sanhuang tablets after intervention;②To clarify the mechanism of Jiangtang Sanhuang tablet in the treatment of T2DM through gut microbiota;③Strengthening gut microbiota-SCFAs-β cell Understanding of the correlation between cell axis and T2DM.Methods1.one Animals were divided into groups;80 SPF grade 6-week-old male T2DM disease model c57bks db/db mice and 10 6-week-old male c57bks dB/M mice in the same nest.After one week of adaptive feeding,all experimental mice were randomly divided into groups by SPSS,and each group was administered daily by gavage.db/m mice in normal group(group n)and db/db mice in model group(group M)(0.5ml D normal saline),low dose group of jiangtangsanhuang tablet(JL group)(jiangtangsanhuang tablet normal saline solution 1.24g/kg d),high dose group of jiangtangsanhuang tablet(JH group)(jiangtangsanhuang tablet normal saline solution 2.48g/kg d),Metformin group(met group)(metformin physiological solution 0.248 g/kg·d);db/db mice in three groups of flora transplantation group(FMT)were fed with 200 μl ABX solution every day for 14 days from the fourth week of the experiment.1g/kg fecal bacterial solution was introduced into FMTa group(JH group mice fecal flora transplantation),FMTb group(M group mice fecal flora transplantation)and FMTc group(N group mice fecal flora transplantation)by enema for 14 days.Mice in each group were fed with normal SPF drinking water and feed for 8 weeks.At the end of the experiment,fasting for 12 hours,taking blood from eyeballs after anesthesia with 4%chloral hydrate,centrifuging the serum,and placing it in the refrigerator at-80℃,The distal colon and pancreatic tissues were quickly removed,and some colon samples were fixed in 2.5%glutaraldehyde solution and stored in a 4℃ refrigerator;Part of it was put in 4%paraformaldehyde solution for histochemical experiment;The remaining colonic and pancreatic tissues were placed in-80℃ refrigerator..2.General condition of mice:weigh the weight of mice every 2 weeks,record the drinking water,food intake and activity state,and measure the fasting blood glucose at the tail tip..3.Glucose metabolism level,insulin secretion and resistance:OGTT and ITT were measured in the last week of the experiment,and the area under the curve(AUC)was calculated;After 8 weeks of the experiment,the serum fins level of mice in each group was measured by INS ELISA kit and the serum Glu level was measured by Glu test kit,and the insulin resistance index and islet were calculated β Cell function index.4.Observation of pancreatic tissue structure and morphology:the number and structural changes of islet mass and islet cells were observed by HE staining.5.16S rDNA amplicon sequencing and SCFAs targeted metabolism detection by GCMS:fecal samples of mice in each group were collected once a day,and 1-2 complete feces were collected for each group for 3 times.The changes of flora structure and flora after transplantation and the content of SCFAs were detected.6.Serum GLP1 content:the serum GLP1 content of mice in each group was measured by mouse GLP1 ELISA kit.7.Ultrastructural observation and protein and gene expression detection of distal colon:at the end of the experiment,take the distal colon and observe the ultrastructural changes of distal colon and the changes of secretory structure and function of L cells under electron microscope;The expression and distribution of GLP1 in distal colon were observed by IHC;The protein and gene expression of GLP1 and GPR43 in intestinal tissue were detected by Western blotting and real time PCR,respectively.Results1 Effects of Jiangtang Sanhuang tablet on blood glucose and islet function in T2DM mice1.1 Effect of db/db on glucose metabolism in miceBefore the experiment,there was no significant difference in fasting blood glucose level between db/db mice in each group,which was significantly higher than that in the normal group(P<0.05).With the progress of the experiment,the blood glucose of db/db mice in each group gradually increased.The fasting blood glucose level of model group increased most significantly in the 8th week of the experiment,which was significantly different from that of Jiangtang Sanhuang tablet low-dose group and metformin control group(P<0.05).OGTT experiment found that except group N,the blood glucose value before gavage could be restored,the blood glucose value before gavage had not been restored in other groups,but the blood glucose value in JH group was significantly lower than that in other drug groups;The blood glucose curve of group M was higher as a whole,and the blood glucose had no downward trend.At 2h,the blood glucose was still much higher than that before gavage.Compared with group N,the blood glucose AUC of db/db mice in the whole group was significantly higher,and there was significant difference in group M(P<0.05).1.2 Insulin resistance,islet function and pathological changes of islet cells in miceIn ITT experiment,after insulin injection,the blood glucose of group N mice showed a gentle downward trend without obvious fluctuation.The blood glucose of db/db mice fluctuated greatly,reached the lowest value of blood glucose in the same group at 1 h after injection,and increased to 0h blood glucose at 2h,even higher than that before injection.Only JH group decreased and fluctuated gently,and the rebound of blood glucose at 2h was small.In the whole group of serum FINS,the content of INS in group N was the lowest,and the ins in dB/db mice was higher than that in WT mice(P<0.05);M group was lower than JH,JL and MET group(P<0.05);Compared with the drug intervention group,the level of INS in JL group was lower,but there was no significant difference among JH,JL and met groups(P>0.05).The serum FBG level was significantly higher than the FBG blood glucose measured at the tail tip.Compared with group M,the blood glucose of JH,JL and met groups after drug intervention decreased significantly,and the blood glucose of JL and met groups was statistically significant(P<0.05);Compared with JH,JL and met groups,the FBG of JL group was lower,but there was no significant difference(P>0.05).HOMA-IR and HOMA-β It is suggested that except the normal group,there are different degrees of insulin resistance and islets β Cell function was damaged,and the model group was decompensated,which was improved in Jiangtang Sanhuang tablet and metformin group.2 Effect of Jiangtang Sanhuang tablet on the structure of gut microbiota in T2DM miceThere were significant differences in the structure of flora between the normal group and the model group,and the diversity of intestinal flora of db/db mice in the model group was higher.Jiangtang Sanhuang tablet does not change the diversity of gut microbiota in mice,but adjusts the structure and produces beneficial bacteria conducive to the metabolism of short chain fatty acids(SCFAs),such as ruminococcaceae and faecalibaculum.After transplantation of pseudosterile mice,the diversity of flora in FMTa group increased and its structure changed,but there was no significant change in FMTc group.In OGTT experiment,FMTa group was lower than FMTb and FMTC groups,but there was no statistical significance.The hypoglycemic effect of FMTa group was worse than that of Jiangtang Sanhuang tablet group,indicating that the effect of oral Jiangtang Sanhuang tablet in adjusting the flora structure was better than that of direct flora transplantation.3 Effect of Jiangtang Sanhuang tablet on SCFAs metabolites in T2DM miceThe content of SCFAs in group M was the lowest,and the total content of SCFAs in high and low dose groups of Jiangtang Sanhuang tablets and metformin group was significantly higher.Acetic acid,propionic acid and butyric acid accounted for the main components of SCFAs in each group.It is related to the production of SCFAs by ruminococcaceae,faecalibaculum and other flora with high abundance in Jiangtang Sanhuang tablets.4 Molecular mechanism of Jiangtang Sanhuang tablet on islet cell protection of intestinal derived GLP1 in T2DM miceTransmission electron microscope observation showed that the distal structure of colon in group M was seriously damaged,and the treatment group was improved in varying degrees.IHC found that GLP1 was mainly expressed on the cell membrane of intestinal columnar epithelium and lamina propria in normal mice.The expression of db/db GLP1 in group M was significantly reduced.After Jiangtang Sanhuang tablet intervention,the expression of GLP1 protein in the intestinal tissue of db/db mice was increased,but not in metformin group.GLP1 and GPR43 in the intestinal tissue of db/db mice in the model group were significantly reduced in RNA and protein levels.The intervention of Jiangtang Sanhuang tablet could increase the expression of GLP1 and GPR43 in the intestinal tissue.Jiangtang Sanhuang tablet may improve the protection of intestinal structural integrity,promote the expression of GPR 43 receptor on L cell membrane and secrete intestinal derived GLP1.Conclusion1.The diversity and structure of intestinal flora were significantly different between T2DM db/db mice and wild-type normal mice.The diversity of gut microbiota,structural disorder and harmful bacteria increased in db/db mice.2.Jiangtang Sanhuang tablet can adjust the structure of gut microbiota of T2DM mice without reducing the diversity of flora.The beneficial bacteria producing SCFAs and the metabolites SCFAs are also increased,mainly acetic acid,propionic acid and butyric acid.3.The structure of distal colon tissue in db/db mice was seriously damaged.Jiangtang Sanhuang tablet could protect the distal colon tissue and improve the number and function of intestinal epithelial L cells.4.Jiangtang Sanhuang tablet can reduce blood sugar,promote insulin secretion and alleviate islet cell damage.By improving the SCFAs mainly composed of acetic acid,propionic acid and butyric acid,it can bind to GPR43 receptor on L cell membrane,improve the secretion ability of L cells and promote the increase of intestinal derived GLP1. |
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